Bottom Line:
In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating.In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression.In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer.

ABSTRACTTumor necrosis factor-alpha, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell-cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. In contrast, ECs failed to express E-selectin when plated on poly-L-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 +/- 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule-1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co- culture of subconfluent ECs with PECAM-1- coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.

Figure 6: The non–cytokine-mediated pathway of E-selectin expression is additive with TNF-induced E-selectin expression and supports neutrophil adherence. (a) E-selectin expression on ECs plated at cobblestone density (105 cells per cm2) or subconfluent densities (0.25 × 105 cells per cm2) 15 h after plating (black). In some groups 1 U/ml of TNF-α was added 4 h before analysis (gray). Results shown are the mean ± SEM of four EC lines. Differences between non–cytokine-induced expression and expression after TNF stimulation, were significant by paired t test (P = 0.04, confluent density ECs and P = 0.02 subconfluent density ECs). (B) 15 h after plating EC at either confluent (solid, 105 cells per cm2) or subconfluent densities (stripes, 0.25 × 105 cells per cm2) neutrophils were added. 30 min later, attached neutrophils were removed by gentle washing and the number of neutrophils adherent per EC were counted using microscopy. A plot of the distribution frequency is shown. 176 ECs at cobblestone density and 168 ECs at subconfluent density were counted in the experiment shown, which is representative of at least three experiments. The two groups were statistically different (P = 0.01) by the Kolmogorov-Smirnov test.

Mentions:
Low doses of TNF-α were able to increase E-selectin expression on both low and high density ECs (Fig. 6 a) suggesting that the cytokine and non-cytokine pathways of induction are at least additive. Although the level of E-selectin expression induced by the cytokine-independent mechanism is less than that induced by TNF or IL-1 (Figs. 5 and 6), it is however, functionally relevant. Assessment of the number of neutrophils adherent per EC showed that there was an increase in the number of neutrophils attached per EC in cells plated at low versus high density EC (Fig. 6 b). In three experiments, the percentage of EC supporting the adhesion of two or more neutrophils was 24.3% ± 14% for low density EC and 1.4% ± 1.0% for high density EC (mean ± SEM; P = 0.02).

Figure 6: The non–cytokine-mediated pathway of E-selectin expression is additive with TNF-induced E-selectin expression and supports neutrophil adherence. (a) E-selectin expression on ECs plated at cobblestone density (105 cells per cm2) or subconfluent densities (0.25 × 105 cells per cm2) 15 h after plating (black). In some groups 1 U/ml of TNF-α was added 4 h before analysis (gray). Results shown are the mean ± SEM of four EC lines. Differences between non–cytokine-induced expression and expression after TNF stimulation, were significant by paired t test (P = 0.04, confluent density ECs and P = 0.02 subconfluent density ECs). (B) 15 h after plating EC at either confluent (solid, 105 cells per cm2) or subconfluent densities (stripes, 0.25 × 105 cells per cm2) neutrophils were added. 30 min later, attached neutrophils were removed by gentle washing and the number of neutrophils adherent per EC were counted using microscopy. A plot of the distribution frequency is shown. 176 ECs at cobblestone density and 168 ECs at subconfluent density were counted in the experiment shown, which is representative of at least three experiments. The two groups were statistically different (P = 0.01) by the Kolmogorov-Smirnov test.

Mentions:
Low doses of TNF-α were able to increase E-selectin expression on both low and high density ECs (Fig. 6 a) suggesting that the cytokine and non-cytokine pathways of induction are at least additive. Although the level of E-selectin expression induced by the cytokine-independent mechanism is less than that induced by TNF or IL-1 (Figs. 5 and 6), it is however, functionally relevant. Assessment of the number of neutrophils adherent per EC showed that there was an increase in the number of neutrophils attached per EC in cells plated at low versus high density EC (Fig. 6 b). In three experiments, the percentage of EC supporting the adhesion of two or more neutrophils was 24.3% ± 14% for low density EC and 1.4% ± 1.0% for high density EC (mean ± SEM; P = 0.02).

Bottom Line:
In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating.In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression.In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer.

ABSTRACTTumor necrosis factor-alpha, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell-cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. In contrast, ECs failed to express E-selectin when plated on poly-L-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 +/- 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule-1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co- culture of subconfluent ECs with PECAM-1- coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.